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Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood.

Zhou H, Martinez H, Sun B, Li A, Zimmer M, Katsanis N, Davis EE, Kurtzberg J, Lipnick S, Noggle S, Rao M, Chang S - Stem Cell Rev (2015)

Bottom Line: The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports.Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way.In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

View Article: PubMed Central - PubMed

Affiliation: The New York Stem Cell Foundation Research Institute, New York, NY, 10032, USA, mzhou@nyscf.org.

ABSTRACT
Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

No MeSH data available.


Related in: MedlinePlus

Cell fate characterization. a, Expression of blood sublineage cell surface markers in PBMC during 4-day cell expansion. b, Expression of blood sublineages and common pluripotency markers in four independent reprogramming cultures RC_A, RC_B, RC_C, RC_D, collected on day 14 after reprogramming induction. c, Flow cytometry analysis of SSEA4 and TRA-1-60 expression on representative reprogramming culture RC_B. d, Flow cytometry analysis of CD71 and TRA-1-60 expression on representative reprogramming culture RC_B. e, Purified iPSCs express pluripotency markers OCT4 and TRA-1-60. DAPI services as a control, f, Flow cytometry analysis of CD71 and CD13 expression on purified iPSCs
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Fig3: Cell fate characterization. a, Expression of blood sublineage cell surface markers in PBMC during 4-day cell expansion. b, Expression of blood sublineages and common pluripotency markers in four independent reprogramming cultures RC_A, RC_B, RC_C, RC_D, collected on day 14 after reprogramming induction. c, Flow cytometry analysis of SSEA4 and TRA-1-60 expression on representative reprogramming culture RC_B. d, Flow cytometry analysis of CD71 and TRA-1-60 expression on representative reprogramming culture RC_B. e, Purified iPSCs express pluripotency markers OCT4 and TRA-1-60. DAPI services as a control, f, Flow cytometry analysis of CD71 and CD13 expression on purified iPSCs

Mentions: As mononuclear cells are a heterogeneous group of nucleated cells, incubation of mononuclear cells with a combination of cytokines could selectively expand one or several types of blood cells. We next asked what cell type(s) are reprogrammed using this protocol by characterizing the cell fates during cell expansion and reprogramming. During the 4-day PBMC expansion period, a portion of mononuclear cells were collected daily for characterization using flow cytometry. At the end of the 4-day cell expansion, an 80-200–fold expansion of CD71+ erythroblast, a ~1.5–fold expansion of CD13+ cell, a slight increase of CD34+ progenitors, and a decrease in the percentage of CD14 (~12 fold decrease) were observed. While >95 % of the cells manifested a hematopoietic cells characteristic (CD45+), this culture condition did not expand mature T-lymphocytes (CD3+) and B-lymphocytes (CD19+), as indicated by decreased and low percentage of cells present in the expanded cell culture (Fig. 3a, 0.21–0.40 % CD3+ and 0.03–0.64 % CD19+). These observations indicated that CD13+ myeloid progenitors and/or CD71+ erythroblast cells are dominant after 4 days of cell expansion.Fig. 3


Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood.

Zhou H, Martinez H, Sun B, Li A, Zimmer M, Katsanis N, Davis EE, Kurtzberg J, Lipnick S, Noggle S, Rao M, Chang S - Stem Cell Rev (2015)

Cell fate characterization. a, Expression of blood sublineage cell surface markers in PBMC during 4-day cell expansion. b, Expression of blood sublineages and common pluripotency markers in four independent reprogramming cultures RC_A, RC_B, RC_C, RC_D, collected on day 14 after reprogramming induction. c, Flow cytometry analysis of SSEA4 and TRA-1-60 expression on representative reprogramming culture RC_B. d, Flow cytometry analysis of CD71 and TRA-1-60 expression on representative reprogramming culture RC_B. e, Purified iPSCs express pluripotency markers OCT4 and TRA-1-60. DAPI services as a control, f, Flow cytometry analysis of CD71 and CD13 expression on purified iPSCs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493720&req=5

Fig3: Cell fate characterization. a, Expression of blood sublineage cell surface markers in PBMC during 4-day cell expansion. b, Expression of blood sublineages and common pluripotency markers in four independent reprogramming cultures RC_A, RC_B, RC_C, RC_D, collected on day 14 after reprogramming induction. c, Flow cytometry analysis of SSEA4 and TRA-1-60 expression on representative reprogramming culture RC_B. d, Flow cytometry analysis of CD71 and TRA-1-60 expression on representative reprogramming culture RC_B. e, Purified iPSCs express pluripotency markers OCT4 and TRA-1-60. DAPI services as a control, f, Flow cytometry analysis of CD71 and CD13 expression on purified iPSCs
Mentions: As mononuclear cells are a heterogeneous group of nucleated cells, incubation of mononuclear cells with a combination of cytokines could selectively expand one or several types of blood cells. We next asked what cell type(s) are reprogrammed using this protocol by characterizing the cell fates during cell expansion and reprogramming. During the 4-day PBMC expansion period, a portion of mononuclear cells were collected daily for characterization using flow cytometry. At the end of the 4-day cell expansion, an 80-200–fold expansion of CD71+ erythroblast, a ~1.5–fold expansion of CD13+ cell, a slight increase of CD34+ progenitors, and a decrease in the percentage of CD14 (~12 fold decrease) were observed. While >95 % of the cells manifested a hematopoietic cells characteristic (CD45+), this culture condition did not expand mature T-lymphocytes (CD3+) and B-lymphocytes (CD19+), as indicated by decreased and low percentage of cells present in the expanded cell culture (Fig. 3a, 0.21–0.40 % CD3+ and 0.03–0.64 % CD19+). These observations indicated that CD13+ myeloid progenitors and/or CD71+ erythroblast cells are dominant after 4 days of cell expansion.Fig. 3

Bottom Line: The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports.Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way.In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

View Article: PubMed Central - PubMed

Affiliation: The New York Stem Cell Foundation Research Institute, New York, NY, 10032, USA, mzhou@nyscf.org.

ABSTRACT
Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

No MeSH data available.


Related in: MedlinePlus